Heteromeric complexes of native collectin kidney 1 and collectin liver 1 are found in the circulation with MASPs and activate the complement system.

The complement system is an important part of the innate immune system. The complement cascade may be initiated downstream of the lectin activation pathway upon binding of mannan-binding lectin, ficolins, or collectin kidney 1 (CL-K1, alias CL-11) to suitable microbial patterns consisting of carbohydrates or acetylated molecules. During purification and characterization of native CL-K1 from plasma, we observed that collectin liver 1 (CL-L1) was copurified. Based on deglycosylation and nonreduced/reduced two-dimensional SDS-PAGE, we detected CL-K1 and CL-L1 in disulfide bridge-stabilized complexes. Heteromeric complex formation in plasma was further shown by ELISA and transient coexpression. Judging from the migration pattern on two-dimensional SDS-PAGE, the majority of plasma CL-K1 was found in complex with CL-L1. The ratio of this complex was in favor of CL-K1, suggesting that a heteromeric subunit is composed of one CL-L1 and two CL-K1 polypeptide chains. We found that the complex bound to mannan-binding lectin-associated serine proteases (MASPs) with affinities in the nM range in vitro and was associated with both MASP-1/-3 and MASP-2 in plasma. Upon binding to mannan or DNA in the presence of MASP-2, the CL-L1-CL-K1 complex mediated deposition of C4b. In favor of large oligomers, the activity of the complex was partly determined by the oligomeric size, which may be influenced by an alternatively spliced variant of CL-K1. The activity of the native heteromeric complexes was superior to that of recombinant CL-K1. We conclude that CL-K1 exists in circulation in the form of heteromeric complexes with CL-L1 that interact with MASPs and can mediate complement activation.

GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.

With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked,

Degradation of the potato glycoalkaloids--alpha-solanine and alpha-chaconine in groundwater.

The potato glycoalkaloids alpha-chaconine and alpha-solanine are produced in high amounts in potato plants from where release to soil takes place. Degradation of the compounds in groundwater was investigated, as their fate in the terrestrial environment is unknown. Abiotic and microbial degradation were followed in groundwater sampled from below a potato field and spiked with the glycoalkaloids (115 nmol/l). Degradation was primarily microbial and the glycoalkaloids were degraded within 21-42 days. The metabolites beta(1)-solanine, gamma-solanine, and solanidine were formed from alpha-solanine, while beta-chaconine, gamma-chaconine and solanidine were detected from alpha-chaconine. Thus, indigenous groundwater microorganisms are capable of degrading the glycoalkaloids.

Potato glycoalkaloids in soil-optimising liquid chromatography-time-of-flight mass spectrometry for quantitative studies.

Potato glycoalkaloids are produced in high amounts in potato fields during the growth season and losses to soil potentially impact shallow groundwater and via tiles to fresh water ecosystems. A quantitative liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) method for determination and quantification of potato glycoalkaloids and their metabolites in aqueous soil extracts was developed. The LC-ESI-TOF-MS method had linearities up to 2000microg/L for alpha-solanine and alpha-chaconine and up to 760microg/L for solanidine. No matrix effect was observed, and the detection limits found were in the range 2.2-4.7microg/L. The method enabled quantification of the potato glycoalkaloids in environmental samples.

The naturally occurring carcinogen ptaquiloside is present in groundwater below bracken vegetation.

The present study demonstrates unequivocally the presence of the natural carcinogen ptaquiloside and its transformation product pterosin B in groundwater and surface water. Groundwater concentrations up to 0.23 nmol/L (92 ng/L) ptaquiloside and up to 2.2 nmol/L (0.47 µg/L) pterosin B were found. Of 21 groundwater samples, 5 contained ptaquiloside, exceeding the estimated threshold for drinking water (1.3-40 pmol/L). The results are critical for water abstraction in bracken-infested areas.

Determination of site-specific glycan heterogeneity on glycoproteins.

The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.

Structural analysis of N- and O-glycans released from glycoproteins.

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive ß-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

Mucin-type O-glycosylation--putting the pieces together.

The O-glycosylation of Ser and Thr by N-acetylgalactosamine-linked (mucin-type) oligosaccharides is often overlooked in protein analysis. Three characteristics make O-linked glycosylation more difficult to analyse than N-linked glycosylation, namely: (a) no amino acid consensus sequence is known; (b) there is no universal enzyme for the release of O-glycans from the protein backbone; and (c) the density and number of occupied sites may be very high. For significant biological conclusions to be drawn, the complete picture of O-linked glycosylation on a protein needs to be determined. This review specifically addresses the analytical approaches that have been used, and the challenges remaining, in the characterization of both the composition and structure of mucin-type O-glycans, and the determination of the occupancy and heterogeneity at each amino acid attachment site.

Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture.

Past proteomic studies of membrane proteins have often been hampered by the low abundance and relatively high hydrophobicity of these proteins. Proteins are often glycosylated, particularly on the extracellular surface of the plasma membrane, and this characteristic was targeted as an enrichment strategy for identifying membrane proteins. Here, we report a strategy for identifying the tissue membrane glycoproteome, which involves (1) Triton X-114 phase partitioning, (2) isolation of glycosylphosphatidylinositol (GPI)-anchored proteins, and (3) glycoprotein capture using lectin affinity or hydrazine chemistry. Surprisingly, the capture of membrane proteins by lectin affinity and hydrazine chemistry resulted in mostly different populations of enriched glycoproteins. Lectins enriched high molecular weight functional membrane proteins with more potential glycosylation such as those involved in signal transduction and cell adhesion. Conversely, hydrazine chemistry isolated a higher proportion of smaller, enzymatic and peripheral membrane proteins such as solute carrier transporters and cytochrome p450s. We have applied our strategy to characterize the rat liver membrane glycoproteome and identified four new predicted GPI-anchored proteins and two that have not previously been seen in the liver. We also identified 424 nonredundant membrane proteins, of which 335 had potential N-linked glycosylation sites.

Fate of toxic potato glycoalkaloids in a potato field.

The toxic glycoalkaloids, alpha-solanine and alpha-chaconine, are present in all parts of the potato plant and are possibly transferred to the terrestrial environment. The amounts of glycoalkaloids in plant, soil, and groundwater were followed in a potato field to investigate their distribution and fate during the season. The amount of glycoalkaloids in the plants was up to 25 kg/ha during maturity and decreased to below 0.63 kg/ha during plant senescence. The glycoalkaloids were detected in the upper soil (up to 0.6 kg/ha); this amount accounted only for a minor fraction of the amount present in the plants. Maximum glycoalkaloid concentration of 2.8 mg/kg dry weight soil was detected in September. Dissipation during winter appeared to be slow because glycoalkaloids were still present in the soil in March. No traces of glycoalkaloids were detected in the groundwater (detection limit 0.2 microg/L). From these results, the leaching potential of the glycoalkaloids is evaluated to be small.

Degradation of the potato glycoalkaloid alpha-solanine in three agricultural soils.

The toxic glycoalkaloids produced by the potato plant (Solanum tuberosum L.) have previously been found in upper soil from a potato field during several months. Further insight into the fate of the glycoalkaloids is needed, as only little information about their degradation in soil is available. Degradation of the glycoalkaloid, alpha-solanine, has been followed for 42d in three agricultural soils with common texture and carbon contents. A similar degradation pattern was found in all soils, and the kinetics was well described by a sum of two first-order equations. Overall, degradation rates for the initial first reaction were in the range 0.22-1.64d(-1). Estimated half-lives were in the range 1.8-4.1d for the three top soils at 15 degrees C; the fastest degradation was observed in the sandy soil. The major proportion of alpha-solanine in the sandy soil was degraded by the fast process, while the proportion was lower for the two other soils. Fast degradation appeared to be related to the presence of low amount of sorbents. Additionally, degradation was followed at 5 degrees C in A- and C-horizon soil from the sandy location, and for both horizons the half-lives were of similar length (4.7-8.7d). For the slow process, degradation rates were in the range 0.000-0.123d(-1), and residuals were still present in all soils and all temperatures at the end of the experiment (d 42). Overall, fast degradation was found in both top- and subsoil even at low temperatures, and the risk for alpha-solanine leaching to the groundwater appears to be low.

Quantification of ptaquiloside and pterosin B in soil and groundwater using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The carcinogenic compound ptaquiloside is produced by bracken fern (Pteridium aquilinum L.). Ptaquiloside can enter the soil matrix and potentially leach to the aquatic environment, and methods for characterizing ptaquiloside content and fate in soil and groundwater are needed. A sensitive detection method has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analyzing ptaquiloside and its transformation product pterosin B. Detection limits are 0.19 microg/L (ptaquiloside) and 0.15 microg/L (pterosin B), which are 300-650 times better than previously published LC-UV methods. Sequential soil extractions are made using 5 mM ammonium acetate for extraction of ptaquiloside, followed by 80% methanol extraction for pterosin B. Groundwater samples are cleaned-up and preconcentrated by a factor of 20 using solid-phase extraction. The LC-MS/MS method enables quantification of ptaquiloside and pterosin B in soil and groundwater samples at environmentally relevant concentrations and delivers a reliable identification because of the structure-specific detection method.

Characterization of the oligomer structure of recombinant human mannan-binding lectin.

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization.

Sorption-controlled degradation kinetics of MCPA in soil.

The relationship between sorption strength and degradation kinetics has been studied for the pesticide MCPA in a sandy top- and subsoil. After adding two types of sorbents (crushed peat and activated carbon) in various amounts to the sandy soils, sorption, desorption, and mineralization of 14C-MCPA were measured. The obtained Freundlich constants (KF) varied between 0.7 and 27.2 mg(1-nF) x L(nF)/kg, and the first-order mineralization rate constants varied between 0.001 and 0.128 d(-1). The results showed an inverse relationship between sorption strength and mineralization. A higher KF value corresponded to a smaller mineralization rate and less mineralization. A correlation coefficient of r2 = 0.934 between the log-transformed Freundlich desorption coefficient (K(F,des)) and the log-transformed mineralization rate constant (k) was obtained. After 7, 14, 22, and 35 days of incubation, soil samples were consecutively extracted by water, methanol, and 5 M NaOH to separate the remaining 14C into 3 different pools. The extractions showed that the mineralization only proceeded from the water extractable pool of MCPA. Thin-layer chromatography revealed a formation of small amounts of metabolites; <7% of initially added 14C was present as other compounds than 14C-MCPA in the water and methanol extractable pools. The study showed mineralization to be strongly correlated with sorption, represented by the desorption coefficient, and hence stresses the significance of bonding strength for estimating pesticide degradation in soil.